Anti-tgf-beta induction of bone cell function and bone growth

ABSTRACT

The invention regards the modulation of TGF-β activity by administering to a subject an antibody that binds to TGF-β, thereby increasing bone growth, bone formation, bone mass and bone strength. The antibody acts to increase osteoblast number and function while at the same time decreasing osteoclast number and function. Such drugs are useful in the treatment of diseases or disorders such as osteoporosis, Paget&#39;s disease, metastatic bone cancer, myeloma bone disease, bone fractures, etc.

This application claims benefit of priority to U.S. ProvisionalApplication Ser. No. 61/172,539, filed Apr. 24, 2009, the entirecontents of which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the fields of molecular biology andmedicine. More particularly, it relates to the fields of bone disease &injury, bone repair, bone implants, bone grafts, periodontal disease andcancer. Specifically, it deals with the use of anti-TGF-β antibodies topromote bone growth and formation, to increase bone mass and strength,and thereby to treat bone diseases such as osteoporosis, as well as bonetrauma and cancers with bone involvement, including multiple myeloma andmyeloma bone disease.

2. Related Art

Over 200 million people worldwide suffer from bone disorders such asosteoporosis, bone fractures, and periodontal (gum) disease (where theteeth loose surrounding bone). Osteoporosis represents a large andrapidly growing health care problem with an unmet medical need fortherapies that stimulate bone formation. Most current drugs forosteoporosis retard bone degradation but do not stimulate bone formationto replace already lost bone. Compounds that stimulate bone formationthus represent an unmet need in the area of bone disease. Osteoporosisis known to affect approximately 100 million people worldwide—35 millionof whom live in the U.S., Western Europe and Japan. Moreover, over 25million individuals suffer bone fractures yearly, 60 million haveperiodontal disease (in which the tooth loosens from the jaw bone), andanother 18 million have other bone disorders such as bone cancer.

Most current therapies for osteoporosis patients focus on prevention ofbone loss, not bone formation. This remains an important considerationas significant morbidity and mortality are associated with prolonged bedrest in the elderly that occurs post bone fracture, particularly thosewho have suffered hip fractures. Complications of bed rest include bloodclots and pneumonia. These complications are recognized and measures areusually taken to avoid them, but these are hardly the best approach totherapy.

Yet another bone-related health issues is bone reconstruction and,specifically, the ability to reconstruct defects in bone tissue thatresult from traumatic injury, as a consequence of cancer or cancersurgery, as a result of a birth defect, or as a result of aging. Thereis a significant need for more frequent orthopedic implants, and cranialand facial bone are particular targets for this type of reconstructiveneed. The availability of new implant materials, e.g., titanium, haspermitted the repair of relatively large defects. Titanium implantsprovide excellent temporary stability across bony defects. However,experience has shown that a lack of viable bone bridging the defect canresult in exposure of the appliance, infection, structural instabilityand, ultimately, failure to repair the defect.

Autologous bone grafts are another possibility to deal with bone injury,but they have several demonstrated disadvantages in that they must beharvested from a donor site such as iliac crest or rib, they usuallyprovide insufficient bone to completely fill the defect, and the bonethat does form is sometimes prone to infection and resorption. Partiallypurified xenogeneic preparations are not practical for clinical usebecause microgram quantities are purified from kilograms of bovine bone,making large scale commercial production both costly and impractical.Allografts and demineralized bone preparations are therefore oftenemployed. Microsurgical transfers of free bone grafts with attached softtissue and blood vessels can close bony defects and allow an immediatesource of blood supply to the graft. However, these techniques are timeconsuming, have been shown to produce a great deal of morbidity, and canonly be used by specially trained individuals.

Another form of bone disease is that resulting from cancer. A number ofcancers metastasize to bone and can result in bone weakening, and someare even associated with bone destruction and bone loss, such as breast,lung, thyroid, kidney and prostate cancer. In addition, Multiple Myelomaand its associated myeloma bone disease (MBD) is not a metastaticcancer. Rather, myeloma cells are derived from the B-cells of the immunesystem that normally reside in the bone marrow and are thereforeintimately associated with bone. Indeed, the bone marrowmicroenvironment plays an important role in the growth, survival andresistance to chemotherapy of the myeloma cells, which, in turn,regulate the increased bone loss associated with this disorder(world-wide-web at multiplemyeloma.org). Over 90% of myeloma patientshave bone involvement, versus 40-60% of cancer patients who have bonemetastasis, and over 80% of these MBD patients have intractable bonepain. Additionally, approximately 30% of myeloma patients havehypercalcemia that is a result of the increased osteolytic activityassociated with this disease (Cavo et al., 2006).

Unlike the osteolysis associated with other bone tumors, the MBD lesionsare unique in that they do not heal or repair, despite the patients'having many years of complete remission (world-wide-web atmultiplemyeloma.org; Terpos et al., 2005). Mechanistically, this seemsto be related to the inhibition and/or loss of the bone-formingosteoblast during disease progression. Indeed, bone marker studies andhistomorphometry indicate that both the bone-resorbing osteoclast andosteoblast activity are increased, but balanced early in the disease,whereas overt MBD shows high osteoclast activity and low osteoblastactivity (world-wide-web at multiplemyeloma.org). Thus, MBD is adisorder in which bone formation and bone loss are uncoupled and wouldbenefit from therapies that both stimulate bone formation and retard itsloss. To date, no such therapies exist. Therefore, there continues to bea need for improved methods of stimulating bone formation and increasingbone strength in vivo to treat bone disease and injury, includingcancer.

SUMMARY OF THE INVENTION

Thus, in accordance with the present invention, there is provided amethod of increasing bone mass and/or volume in a subject comprising (a)identifying a patient in need of increased bone mass and/or volume; and(b) administering to said subject an antibody that binds immunologicallythe TGF-β. The antibody may bind all three isoforms of TGF-β. Theantibody may be designated as 1D11, and may be a humanized version of1D11, or any engineered version containing the CDRs of 1D11 in aheterologous framework.

In one embodiment, the antibody is such that it specifically binds to atleast one isoform of TGF-β. In particular embodiments, the anti-TGF-βantibody specifically binds at least one isoform of TGF-β selected fromthe group consisting of TGF-β1, TGF-β2, and TGF-β3. In yet otherembodiments, the anti-TGF-β antibody specifically binds to at least :(a) TGF-β1, TGF-β2, and TGF-β3 (also referred to as “pan-neutralizingantibody”); (b) TGF-β1 and TGF-β2; (c) TGF-β1 and TGF-β3; and (d) TGF-β2and TGF-β3. In various embodiments, the affinity constant Ka of theTGF-β antibody for at least one isoform of TGF-b, which it specificallybinds, is preferably greater than 10⁶ M⁻¹, 10⁷ M⁻¹, 10⁸ M⁻¹, 10⁹ M⁻¹,10¹⁰ M⁻¹, 10¹¹ M⁻¹, or 10¹² M⁻¹. In yet further embodiments, theantibody of the invention specifically binds to a protein substantiallyidentical to human TGF-β1, TGF-β2, and/or TGF-β3. Also contemplated foruse in humans are chimeric and humanized forms and derivatives ofnonhuman antibodies. Producing such variants is well within the ordinaryskill of an artisan (see, e.g., Antibody Engineering, ed. Borrebaeck,2nd ed., Oxford University Press, 1995).

In one embodiment, the anti-TGF-β antibody is a murine monoclonalantibody 1D11 produced by the hybridoma 1D11.16 (ATCC DepositDesignation No. HB 9849, also described in U.S. Pat. Nos. 5,571,714,5,772,998 and 5,783,185). The 1D11 antibody specifically binds all threemammalian isoforms of TGF-β. The sequence of the 1D11 heavy chainvariable region is available under Accession No. AAB46787. In relatedembodiments, the anti-TGF-β antibody is a derivative of 1D11, e.g., anantibody comprising the CDR sequences identical to those in 1D11 (e.g.,a chimeric, humanized or CDR-grafted antibody). In yet a furtherembodiment, the anti-TGF-β antibody is a fully human recombinantantibody.

The antibody may be administered to said subject systemically,intravenously, intra-peritoneally, intramuscularly, subcutaneously ortopically. The antibody may be administered to a bone target site,including injection at the site. The antibody also may be comprised in atime-release device implanted at the site.

The subject may be a human or a non-human animal, such as a mouse, arat, a rabbit, a dog, a cat, a horse, a monkey or a cow. The subject mayhave cancer, or may not. The method may further comprise at least asecond administration of said antibody, including regimens of threeadministrations per week. The subject may receive at least 9administrations. The subject may suffer from osteoporosis, bonefracture, bone loss due to trauma, or Paget's Disease, or from bone lossdue to cancer metastasis. The method may further comprise assessing bonemass following administration of said antibody, such as by bone imaging.

In another embodiment, there is provided a method of increasing bonegrowth in a subject comprising administering to said subject an antibodythat binds immunologically the TGF-β. The antibody may bind all threeisoforms of TGF-β. The antibody may be designated as 1D11, and may be ahumanized version of 1D11, or any engineered version containing the CDRsof 1D11 in a heterologous framework. The antibody may be administered tosaid subject systemically, intravenously, intra-peritoneally,intramuscularly, subcutaneously or topically. The antibody may beadministered to a bone target site, including injection at the site. Theantibody also may be comprised in a time-release device implanted at thesite. The subject may be a human or a non-human animal, such as a mouse,a rat, a rabbit, a dog, a cat, a horse, a monkey or a cow. The subjectmay have cancer, or may not.

In other embodiments, there are provided:

-   -   a method of increasing osteoblast number in a subject comprising        administering to said subject an antibody that binds        immunologically the TGF-β;    -   a method of decreasing osteoclast number in a subject comprising        administering to said subject an antibody that binds        immunologically the TGF-β;    -   a method of increasing bone strength in a subject comprising        administering to said subject an antibody that binds        immunologically the TGF-β; and    -   a method of decreasing TGF-β signaling in a subject comprising        administering to said subject an antibody that binds        immunologically the TGF-β.

It is contemplated that any method or composition described herein canbe implemented with respect to any other method or composition describedherein.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method or composition of theinvention, and vice versa. Furthermore, compositions and kits of theinvention can be used to achieve methods of the invention.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1—Treament regimen. C57B1/6 mice (n=5) were treated i.p. with 1D11or control antibody (13C4) 3 times/week for 28 days. Skeletal parameterswere determined by μCT scanning, histomorphometric analysis andbiochemical testing. Effects of anti-TGF-β therapy on cell distributionand gene/protein expression were also determined.

FIGS. 2A-F—1D11 treatment increases bone volume. 3-dimensional renderedimages from μCT scanned regions of tibial metaphysis (200 μm) showedincreased bone volume in 1D11-treated mice (FIGS. 2A-B), accompanied byan increased BV/TV (FIG. 2C) and BMD (FIG. 2D). A thickening oftrabeculae (FIG. 2E), and decreased trabecular spacing was also observedwithin this region in 1D11-treated animals (FIG. 2F; *=p<0.05).

FIGS. 3A-B—Histomorphometric analysis. Tissue sections of undecalcifiedlumbar vertebrate from animals treated with 1D11 showed a significantincrease in BV/TV and positive changes in trabecular parameters,compared to control animals (FIG. 3A, Von Kossa/Van Gieson, *=p<0.05).Long bone volume was also dramatically increased (FIG. 3B, H&E).

FIGS. 4A-D—Cellular distribution of bone cells following 1D11 treatment.Decalcified tibial sections stained for TRAP showed a considerabledecrease in positively-stained osteoclasts lining the trabecular bonesurface in treated animals compared to controls (FIG. 4A, original magx4; FIG. 4B, original mag x20; black arrows=osteoclasts). Cellulardistribution was quantified by histomorphometric analysis of H&E- andTRAP-stained sections, and demonstrated a significant decrease inosteoclast number and surface on the bone of 1D11-treated mice (FIG. 4C,**p<0.01). Osteoblast numbers were significantly elevated in micetreated with the 1D11 antibody (FIG. 4D, *p<0.05).

FIG. 5—Biomechanical testing. Fresh dissected femurs isolated from 1D11treated or control mice were examined biomechanically by 3-pointbending. Femurs were positioned horizontally and monotonically loaded ata rate of 3 mm/min at the mid-diaphysis. 1D11 treatment significantlyincreased bending strength, yield strength and tissue modulus(**=p<0.01; ***=p<0.001).

DETAILED DESCRIPTION OF THE INVENTION

TGF-β is an abundant bone matrix protein which influences the formation,function and cell-cell interactions of osteoblasts and osteoclasts, tocontrol bone remodeling and maintain adequate bone mass. As such, theTGF-β signaling pathway represents a unique pharmacological target, withthe potential to regulate bone volume and quality through the control ofboth osteoblasts and osteoclasts. Previous studies using murine modelscontaining genetic modifications in the TGF-β signaling pathway haveshown that reduced TGF-β signaling enhances the mechanical propertiesand mineral concentration of the bone matrix, as well as bone mass.Although small molecule inhibitors of TGF-β signaling have been shown todecrease cancer growth and invasiveness, the direct effect of TGF-βblockade on the normal bone marrow environment in non-tumor bearing micehas not been fully addressed.

Therefore, the inventors examined the ability of an anti-TGF-β antibodyto block TGF-β signaling pathways. This antibody positively regulatedosteoblast numbers while simultaneously decreasing the amount of activeosteoclasts in the marrow. This resulted in a profound increase in bonevolume and quality. These findings clearly illustrate the potential ofcompounds which can specifically target the TGF-β signaling pathway invivo, and suggest a therapeutic approach to increase bone mass andstrength. These and other aspects of the invention are set forth indetail below.

I. ANTI-TGF-β

A. TGF-β

Transforming growth factor β (TGF-β) controls proliferation, cellulardifferentiation, and other functions in most cells. It plays a role inimmunity, cancer, heart disease, diabetes, and Marfan syndrome. TGF-βacts as an antiproliferative factor in normal epithelial cells and atearly stages of oncogenesis. Some cells secrete TGF-β, and also havereceptors for TGF-β. This is known as autocrine signalling. Cancerouscells increase their production of TGF-β, which also acts on surroundingcells.

TGF-β is a secreted protein that exists in three isoforms called TGF-β1,TGF-β2 and TGF-β3. It was also the original name for TGF-β1, which wasthe founding member of this family. The TGF-β family is part of asuperfamily of proteins known as the transforming growth factor betasuperfamily, which includes inhibins, activin, anti-müllerian hormone,bone morphogenetic protein, decapentaplegic and Vg-1.

The peptide structures of the three members of the TGF-β family arehighly similar. They are all encoded as large protein precursors; TGF-β1contains 390 amino acids and TGF-β2 and TGF-β3 each contain 412 aminoacids. They each have an N-terminal signal peptide of 20-30 amino acidsthat they require for secretion from a cell, a pro-region (calledlatency associated peptide or LAP), and a 112-114 amino acid C-terminalregion that becomes the mature TGF-β molecule following its release fromthe pro-region by proteolytic cleavage. The mature TGF-β proteindimerizes to produce a 25 KDa active molecule with many conservedstructural motifs. TGF-β has nine cysteine residues that are conservedamong its family; eight form disulfide bonds within the molecule tocreate a cysteine knot structure characteristic of the TGF-β superfamilywhile the ninth cysteine forms a bond with the ninth cysteine of anotherTGF-β molecule to produce the dimer. Many other conserved residues inTGF-β are thought to form secondary structure through hydrophobicinteractions. The region between the fifth and sixth conserved cysteineshouses the most divergent area of TGF-β molecules that is exposed at thesurface of the molecule and is implicated in receptor binding andspecificity of TGF-β.

TGF-β induces apoptosis in numerous cell types. TGF-β can induceapoptosis in two ways: through the SMAD pathway or the DAXX pathway. TheSMAD pathway is the canonical signaling pathway that TGF-β familymembers signal through. In this pathway, TGF-β dimers bind to a type IIreceptor which recruits and phosphorylates a type I receptor. The type Ireceptor then recruits and phosphorylates a receptor regulated SMAD(R-SMAD). SMAD3, an R-SMAD, has been implicated in inducing apoptosis.The R-SMAD then binds to the common SMAD (coSMAD) SMAD4 and forms aheterodimeric complex. This complex then enters the cell nucleus whereit acts as a transcription factor for various genes, including those toactivate the mitogen-activated protein kinase 8 pathway, which triggersapoptosis. TGF-β may also trigger apoptosis via the death associatedprotein 6 (DAXX adapter protein). DAXX has been shown to associate withand bind to the type II TGF-β receptor kinase. TGF-β is believed to beimportant in regulation of the immune system by CD25+ regulatory. TGF-βappears to block the activation of lymphocytes and monocyte derivedphagocytes.

B. Antibodies

Production Methods. In another aspect, the present inventioncontemplates an antibody that is immunoreactive with TGF-β, includingbeing cross-reactive with TGF-β isoforms 1-3. The antibody can be amonoclonal antibody, but use of a polyclonal antibody preparation withthe same TGF-β1-3 specificity could be employed. Means for preparing andcharacterizing antibodies are well known in the art (see, e.g., Harlowand Lane, 1988).

Briefly, polyclonal antibodies are prepared by immunizing an animal withan immunogen (i.e., TGF-β or a fragment thereof) and collecting antiserafrom that immunized animal. A wide range of animal species can be usedfor the production of antisera. Typically, an animal used for productionof antisera is a non-human animal including rabbits, mice, rats,hamsters, pigs or horses. Because of the relatively large blood volumeof rabbits, a rabbit is a preferred choice for production of polyclonalantibodies.

As is well known in the art, a given composition may vary in itsimmunogenicity. It is often necessary therefore to boost the host immunesystem, as may be achieved by coupling a peptide or polypeptideimmunogen to a carrier. Exemplary and preferred carriers are keyholelimpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albuminssuch as ovalbumin, mouse serum albumin or rabbit serum albumin can alsobe used as carriers. Means for conjugating a polypeptide to a carrierprotein are well known in the art and include glutaraldehyde,m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide andbis-biazotized benzidine.

As also is well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Exemplary andpreferred adjuvants include complete Freund's adjuvant (a non-specificstimulator of the immune response containing killed Mycobacteriumtuberculosis), incomplete Freund's adjuvants and aluminum hydroxideadjuvant.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of routes can be used to administer theimmunogen (subcutaneous, intramuscular, intradermal, intravenous andintraperitoneal). The production of polyclonal antibodies may bemonitored by sampling blood of the immunized animal at various pointsfollowing immunization. A second, booster, injection may also be given.The process of boosting and titering is repeated until a suitable titeris achieved. When a desired level of immunogenicity is obtained, theimmunized animal can be bled and the serum isolated and stored, and/orthe animal can be used to generate mAbs.

MAbs may be readily prepared through use of well-known techniques, suchas those exemplified in U.S. Pat. No. 4,196,265, incorporated herein byreference. Typically, this technique involves immunizing a suitableanimal with a selected immunogen composition, i.e., a purified orpartially purified TGF-β protein, polypeptide or peptide or cellexpressing high levels of TGF-β. The immunizing composition isadministered in a manner effective to stimulate antibody producingcells. Rodents such as mice and rats are preferred animals, however, theuse of rabbit, sheep frog cells is also possible. The use of rats mayprovide certain advantages (Goding, 1986), but mice are preferred, withthe BALB/c mouse being most preferred as this is most routinely used andgenerally gives a higher percentage of stable fusions.

Following immunization, somatic cells with the potential for producingantibodies, specifically B-lymphocytes (B-cells), are selected for usein the mAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Spleen cells and peripheral blood cells are preferred, theformer because they are a rich source of antibody-producing cells thatare in the dividing plasmablast stage, and the latter because peripheralblood is easily accessible. Often, a panel of animals will have beenimmunized and the spleen of animal with the highest antibody titer willbe removed and the spleen lymphocytes obtained by homogenizing thespleen with a syringe. Typically, a spleen from an immunized mousecontains approximately 5×10⁷ to 2×10⁸ lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render then incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art (Goding, 1986; Campbell, 1984). For example, wherethe immunized animal is a mouse, one may use P3-X63/Ag8, P3-X63-Ag8.653,NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 andS194/5XX0 Bul; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all usefulin connection with cell fusions.

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1to about 1:1, respectively, in the presence of an agent or agents(chemical or electrical) that promote the fusion of cell membranes.Fusion methods using Sendai virus have been described (Kohler andMilstein, 1975; 1976), and those using polyethylene glycol (PEG), suchas 37% (v/v) PEG, by Gefter et al. (1977). The use of electricallyinduced fusion methods is also appropriate (Goding, 1986).

Fusion procedures usually produce viable hybrids at low frequencies,around 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, unfusedcells (particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryand preferred agents are aminopterin, methotrexate, and azaserine.Aminopterin and methotrexate block de novo synthesis of both purines andpyrimidines, whereas azaserine blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium).Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B-cells can operate this pathway, but they have a limited life spanin culture and generally die within about two weeks. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B-cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired reactivity. The assay should besensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

The selected hybridomas would then be serially diluted and cloned intoindividual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide mAbs. The cell lines may be exploitedfor mAb production in two basic ways. A sample of the hybridoma can beinjected (often into the peritoneal cavity) into a histocompatibleanimal of the type that was used to provide the somatic and myelomacells for the original fusion. The injected animal develops tumorssecreting the specific monoclonal antibody produced by the fused cellhybrid. The body fluids of the animal, such as serum or ascites fluid,can then be tapped to provide mAbs in high concentration. The individualcell lines could also be cultured in vitro, where the mAbs are naturallysecreted into the culture medium from which they can be readily obtainedin high concentrations. mAbs produced by either means may be furtherpurified, if desired, using filtration, centrifugation and variouschromatographic methods such as HPLC or affinity chromatography.

Modified Antibodies. In one embodiment, antibody molecules will comprisefragments (such as (F(ab′), F(ab′)2) that are produced, for example, bythe proteolytic cleavage of the mAbs, or single-chain immunoglobulinsproducible, for example, via recombinant means. Such antibodyderivatives are monovalent. In one embodiment, such fragments can becombined with one another, or with other antibody fragments or receptorligands to form “chimeric” binding molecules. Significantly, suchchimeric molecules may contain substituents capable of binding todifferent epitopes of the same molecule.

It may desirable to “humanize” antibodies produced in non-human hosts inorder to attenuate any immune reaction when used in human therapy. Suchhumanized antibodies may be studied in an in vitro or an in vivocontext. Humanized antibodies may be produced, for example by replacingan immunogenic portion of an antibody with a corresponding, butnon-immunogenic portion (i.e., chimeric antibodies). PCT ApplicationPCT/US86/02269; EP Application 184,187; EP Application 171,496; EPApplication 173,494; PCT Application WO 86/01533; EP Application125,023; Sun et al. (1987); Wood et al. (1985); and Shaw et al. (1988);all of which references are incorporated herein by reference. Generalreviews of “humanized” chimeric antibodies are provided by Morrison(1985; also incorporated herein by reference. “Humanized” antibodies canalternatively be produced by CDR or CEA substitution. Jones et al.(1986); Verhoeyan et al. (1988); Beidler et al. (1988); all of which areincorporated herein by reference.

In more specific embodiments, one may use the CDR regions from antibody1D11 and place these into the framework regions of any other suitableantibody, human or non-human. The anti-TGF-β antibody 1D11 is a murinemonoclonal antibody produced by the hybridoma 1D11.16 (ATCC DepositDesignation No. HB 9849, also described in U.S. Pat. Nos. 5,571,714,5,772,998 and 5,783,185, incorporated by reference herein). The 1D11antibody specifically binds all three mammalian isoforms of TGF-β. Thesequence of the 1D11 heavy chain variable region is available underAccession No. AAB46787.

In related embodiments, the anti-TGF-β antibody is a derivative of 1D11,e.g., an antibody comprising the CDR sequences identical to those in1D11 (e.g., a chimeric, humanized or CDR-grafted antibody). In yet afurther embodiment, the anti-TGF-β antibody is a fully human recombinantantibody.

II. BONE STRUCTURE AND PHYSIOLOGY

Bone is a living, growing tissue. It is porous and mineralized, and madeup of cells, vessels, organic matrix and inorganic hydroxyapatitecrystals. The human skeleton is actually made up of 2 types of bones:the cortical bone and the trabecular bone. Cortical bone representsnearly 80% of the skeletal mass. Cortical bone has a slow turnover rateand a high resistance to bending and torsion. It provides strength wherebending would be undesirable as in the middle of long bones. Trabecularbone only represents 20% of the skeletal mass, but 80% of the bonesurface. It is less dense, more elastic and has a higher turnover ratethan cortical bone.

A. Bone Forming Cells

Osteoprogenitors. Human bone precursor cells are characterized assmall-sized cells that express low amounts of bone proteins(osteocalcin, osteonectin, and alkaline phosphatase) and have a lowdegree of internal complexity (Long et al., 1995). When stimulated todifferentiate, these preosteoblast cells become osteoblast in theirappearance, size, antigenic expression, and internal structure. Althoughthese cells are normally present at very low frequencies in bone marrow,a process for isolating these cells has been described (Long et al.,1995). U.S. Pat. No. 5,972,703 further describes methods of isolatingand using bone precursor cells, and is specifically incorporated hereinby reference.

A number of studies indicate that bone marrow derived cells haveosteogenic potential. The majority of these investigations point tomesenchymal stem cells (MSC) as undergoing differentiation intoosteoblasts when cultured in the presence of bone-active cytokines(Jaiswal et al., 2000; Phinney et al., 1999; Aubin, 1998; Zohar et al.,1997). Mesenchymal stem cells are a pluripotent population capable ofgenerating multiple stromal cell lineages. MSC, as currently used, are aheterogeneous population of cells isolated by plastic adherence, andpropagated by low-density passage. Nonetheless, a recent publicationindicates the clonal nature of cell fate outcomes in MSC indicating thata single MSC cell can give rise to two or three mesenchymal lineages oneof which is usually bone cells (Pittenger et al., 1999). These studiesare consistent with earlier reports that demonstrated the osteogenicpotential of bone marrow stromal cells, in particular the so-calledCFU-f from both mice and human (Friedenstein et al., 1968; Reddi andHuggins, 1972; Friedenstein et al., 1982; Ashton et al., 1985; Bleiberg,1985; Gronthos et al., 1994; Gronthos et al., 1999).

Single-cell isolation of human MSC generated clones that express thesame surface phenotype as unfractionated MSC (Pittenger et al., 1999).Interestingly, of the 6 MSC clones evaluated, 2 retained osteogenic,chrondrogenic and adipogenic potential; others were bipotent (eitherosteo- plus chondrogenic potential, or osteo-adipocytic potential) orwere uni-lineage (chondrocyte). This suggests that MSC themselves areheterogeneous in nature (although culture conditions also may have ledto loss of lineage potential). To date, the self-renewal capacity of MSCremains in question. Nonetheless, these in vitro studies and other invivo studies (Kadiyala et al., 1997; Petite et al., 2000; Krebsbach etal., 1999) show that MSC can commit to the bone cell lineage and developto the state of matrix mineralization in vitro, or bone formation invivo.

Preosteoblasts. Preosteoblasts are intermediate between osteoprogenitorcells and osteoblasts. They show increasing expression of bonephenotypic markers such as alkaline phosphatase (Kale et al., 2000).They have a more limited proliferative capacity, but nonethelesscontinue to divide and produce more preosteoblasts or osteoblasts.

Osteoblasts. An osteoblast is a mononucleate cell that is responsiblefor bone formation. Osteoblasts produce osteoid, which is composedmainly of Type I collagen. Osteoblasts are also responsible formineralization of the osteoid matrix. Bone is a dynamic tissue that isconstantly being reshaped by osteoblasts, which build bone, andosteoclasts, which resorb bone. Osteoblast cells tend to decrease innumber and activity as individuals become elderly, thus decreasing thenatural renovation of the bone tissue.

Osteoblasts arise from osteoprogenitor cells located in the periosteumand the bone marrow. Osteoprogenitors are immature progenitor cells thatexpress the master regulatory transcription factor Cbfa1/Runx2.Osteoprogenitors are induced to differentiate under the influence ofgrowth factors, in particular the bone morphogenetic proteins (BMPs).Aside from BMPs, other growth factors including fibroblast growth factor(FGF), platelet-derived growth factor (PDGF), transforming growth factorβ (TGF-β) may promote the division of osteoprogenitors and potentiallyincrease osteogenesis. Once osteoprogenitors start to differentiate intoosteoblasts, they begin to express a range of genetic markers includingOsterix, Col1, ALP, osteocalcin, osteopontin, and osteonectin. Althoughthe term osteoblast implies an immature cell type, osteoblasts are infact the mature bone cells entirely responsible for generating bonetissue in animals and humans.

Osteoclasts. An osteoclast is a type of bone cell that removes bonetissue by removing its mineralized matrix. This process is known as boneresorption. Osteoclasts and osteoblasts are instrumental in controllingthe amount of bone tissue: osteoblasts form bone, osteoclasts resorbbone. Osteoclasts are formed by the fusion of cells of themonocyte-macrophage cell lineage. Osteoclasts are characterized by highexpression of tartrate resistant acid phosphatase (TRAP) and cathepsinK.

Osteoclast formation requires the presence of RANK ligand (receptoractivator of nuclear factor κβ) and M-CSF (Macrophage colony-stimulatingfactor). These membrane bound proteins are produced by neighbouringstromal cells and osteoblasts; thus requiring direct contact betweenthese cells and osteoclast precursors. M-CSF acts through its receptoron the osteoclast, c-fms (colony stimulating factor 1 receptor), atransmembrane tyrosine kinase-receptor, leading to secondary messengeractivation of tyrosine kinase Src. Both of these molecules are necessaryfor osteoclastogenesis and are widely involved in the differentiation ofmonocyte/macrophage derived cells. RANKL is a member of the tumornecrosis family (TNF), and is essential in osteoclastogenesis. RANKLknockout mice exhibit a phenotype of osteopetrosis and defects of tootheruption, along with an absence or deficiency of osteoclasts. RANKLactivates NF-κβ (nuclear factor-κβ) and NFATc1 (nuclear factor ofactivated t cells, cytoplasmic, calcineurin-dependent 1) through RANK.NF-κβ activation is stimulated almost immediately after RANKL-RANKinteraction occurs, and is not upregulated. NFATc1 stimulation, however,begins ˜24-48 hours after binding occurs and its expression has beenshown to be RANKL dependent. Osteoclast differentiation is inhibited byosteoprotegerin (OPG), which binds to RANKL thereby preventinginteraction with RANK.

B. Bone Formation

The formation of bone during the fetal stage of development occurs bytwo processes: intramembranous ossification and endochondralossification. Intramembranous ossification mainly occurs duringformation of the flat bones of the skull; the bone is formed frommesenchyme tissue. The steps in intramembranous ossification aredevelopment of ossification center, calcification, formation oftrabeculae and development of periosteum. Endochondral ossification, onthe other hand, occurs in long bones, such as limbs; the bone is formedaround a cartilage template. The steps in endochondral ossification aredevelopment of cartilage model, growth of cartilage model, developmentof the primary ossification center and development of the secondaryossification center.

Endochondral ossification begins with points in the cartilage called“primary ossification centers.” They mostly appear during fetaldevelopment, though a few short bones begin their primary ossificationafter birth. They are responsible for the formation of the diaphyses oflong bones, short bones and certain parts of irregular bones. Secondaryossification occurs after birth, and forms the epiphyses of long bonesand the extremities of irregular and flat bones. The diaphysis and bothepiphyses of a long bone are separated by a growing zone of cartilage(the epiphyseal plate). When the child reaches skeletal maturity (18 to25 years of age), all of the cartilage is replaced by bone, fusing thediaphysis and both epiphyses together (epiphyseal closure).

Remodeling or bone turnover is the process of resorption followed byreplacement of bone with little change in shape and occurs throughout aperson's life. Osteoblasts and osteoclasts, coupled together viaparacrine cell signalling, are referred to as bone remodeling units. Thepurpose of remodeling is to regulate calcium homeostasis, repairmicro-damaged bones (from everyday stress) but also to shape andsculpture the skeleton during growth.

The process of bone resorption by the osteoclasts releases storedcalcium into the systemic circulation and is an important process inregulating calcium balance. As bone formation actively fixes circulatingcalcium in its mineral form, removing it from the bloodstream,resorption actively unfixes it thereby increasing circulating calciumlevels. These processes occur in tandem at site-specific locations.

Repeated stress, such as weight-bearing exercise or bone healing,results in the bone thickening at the points of maximum stress (Wolff slaw). It has been hypothesized that this is a result of bone'spiezoelectric properties, which cause bone to generate small electricalpotentials under stress.

III. TREATMENTS

A. Bone Diseases and Conditions

There is a plethora of conditions which are characterized by the need toenhance bone formation or to inhibit bone resorption and thus wouldbenefit from the use of anti-TGF-β antibodies or combinations ofanti-TGF-β antibodies and second agents as described above or cellstreated therewith in promoting bone formation and/or bone repair.Perhaps the most obvious is the case of bone fractures, where it wouldbe desirable to stimulate bone growth and to hasten and complete bonerepair. Agents that enhance bone formation would also be useful infacial reconstruction procedures. Other bone deficit conditions includebone segmental defects, periodontal disease, metastatic bone disease,osteolytic bone disease and conditions where connective tissue repairwould be beneficial, such as healing or regeneration of cartilagedefects or injury. Also of great significance is the chronic conditionof osteoporosis, including age-related osteoporosis and osteoporosisassociated with post-menopausal hormone status. Other conditionscharacterized by the need for bone growth include primary and secondaryhyperparathyroidism, disuse osteoporosis, diabetes-related osteoporosis,and glucocorticoid-related osteoporosis. Several other conditions, suchas, for example, vitamin D deficiency, exists.

Fracture. The first example is the otherwise healthy individual whosuffers a fracture. Often, clinical bone fracture is treated by castingto alleviate pain and allow natural repair mechanisms to repair thewound. There has been progress in the treatment of fracture in recenttimes, however, even without considering the various complications thatmay arise in treating fractured bones, any new procedures to increasebone healing in normal circumstances would represent a great advance.

Periodontal Disease. Progressive periodontal disease leads to tooth lossthrough destruction of the tooth's attachment to the surrounding bone.Approximately 5-20% of the U.S. population (15-60 million individuals)suffers from severe generalized periodontal disease, and there are 2million related surgical procedures. Moreover, if the disease is definedas the identification of at least one site of clinical attachment loss,then approximately 80% of all adults are affected, and 90% of those aged55 to 64 years. If untreated, approximately 88% of affected individualsshow moderate to rapid progression of the disease' which shows a strongcorrelation with age. The major current treatment for periodontaldisease is regenerative therapy consisting of replacement of lostperiodontal tissues. The lost bone is usually treated with anindividual's own bone and bone marrow, due to their high osteogenicpotential. Bone allografts (between individuals) can also be performedusing stored human bone. Although current periodontal cost analyses arehard to obtain, the size of the affected population and the current useof bone grafts as a first-order therapy strongly suggest that this arearepresents an attractive target for bone-building therapies.

Osteopenia/osteoporosis. The terms osteopenia and osteoporosis refers toa heterogeneous group of disorders characterized by decreased bone massand fractures. Osteopenia is a bone mass that is one or more standarddeviations below the mean bone mass for a population; osteoporosis isdefined as 2.5 SD or lower. An estimated 20-25 million people are atincreased risk for fracture because of site-specific bone loss. Riskfactors for osteoporosis include increasing age, gender (more females),low bone mass, early menopause, race (Caucasians in general; asian andhispanic females), low calcium intake, reduced physical activity,genetic factors, environmental factors (including cigarette smoking andabuse of alcohol or caffeine), and deficiencies in neuromuscular controlthat create a propensity to fall.

More than a million fractures in the U.S. each year can be attributed toosteoporosis. In economic terms, the costs (exclusive of lost wages) forosteoporosis therapies are $35 billion worldwide. Demographic trends(i.e., the gradually increasing age of the U.S. population) suggest thatthese costs may increase to $62 billion by the year 2020. Clearly,osteoporosis is a significant health care problem.

Osteoporosis, once thought to be a natural part of aging among women, isno longer considered age or gender-dependent. Osteoporosis is defined asa skeletal disorder characterized by compromised bone strengthpredisposing to an increased risk of fracture. Bone strength reflectsthe integration of two main features: bone density and bone quality.Bone density is expressed as grams of mineral per area or volume and inany given individual is determined by peak bone mass and amount of boneloss. Bone quality refers to architecture, turnover, damage accumulation(e.g., microfractures) and mineralization. A fracture occurs when afailure-inducing force (e.g., trauma) is applied to osteoporotic bone.

Current therapies for osteoporosis patients focus on fractureprevention, not for promoting bone formation or fracture repair. Thisremains an important consideration because of the literature, whichclearly states that significant morbidity and mortality are associatedwith prolonged bed rest in the elderly, particularly those who havesuffered hip fractures. Complications of bed rest include blood clotsand pneumonia. These complications are recognized and measures areusually taken to avoid them, but these is hardly the best approach totherapy. Thus, the osteoporotic patient population would benefit fromnew therapies designed to strengthen bone and speed up the fracturerepair process, thus getting these people on their feet before thecomplications arise.

Bone Reconstruction/Grafting. A fourth example is related to bonereconstruction and, specifically, the ability to reconstruct defects inbone tissue that result from traumatic injury; as a consequence ofcancer or cancer surgery; as a result of a birth defect; or as a resultof aging. There is a significant need for more frequent orthopedicimplants, and cranial and facial bone are particular targets for thistype of reconstructive need. The availability of new implant materials,e.g., titanium, has permitted the repair of relatively large defects.Titanium implants provide excellent temporary stability across bonydefects and are an excellent material for bone implants or artificialjoints such as hip, knee and joint replacements. However, experience hasshown that a lack of viable bone binding to implants the defect canresult in exposure of the appliance to infection, structural instabilityand, ultimately, failure to repair the defect. Thus, a therapeutic agentthat stimulates bone formation on or around the implant will facilitatemore rapid recovery.

Autologous bone grafts are another possibility, but they have severaldemonstrated disadvantages in that they must be harvested from a donorsite such as iliac crest or rib, they usually provide insufficient boneto completely fill the defect, and the bone that does form is sometimesprone to infection and resorption. Partially purified xenogeneicpreparations are not practical for clinical use because microgramquantities are purified from kilograms of bovine bone, making largescale commercial production both costly and impractical. Allografts anddemineralized bone preparations are therefore often employed, but sufferfrom their devitalized nature in that they only function as scaffoldsfor endogenous bone cell growth.

Microsurgical transfers of free bone grafts with attached soft tissueand blood vessels can close bony defects with an immediate source ofblood supply to the graft. However, these techniques are time consuming,have been shown to produce a great deal of morbidity, and can only beused by specially trained individuals. Furthermore, the bone implant isoften limited in quantity and is not readily contoured. In the mandible,for example, the majority of patients cannot wear dental appliancesusing presently accepted techniques (even after continuity isestablished), and thus gain little improvement in the ability tomasticate.

In connection with bone reconstruction, specific problem areas forimprovement are those concerned with treating large defects, such ascreated by trauma, birth defects, or particularly, following tumorresection; and also the area of artificial joints. The success oforthopaedic implants, interfaces and artificial joints could conceivablybe improved if the surface of the implant, or a functional part of animplant, were to be coated with a bone stimulatory agent. The surface ofimplants could be coated with one or more appropriate materials in orderto promote a more effective interaction with the biological sitesurrounding the implant and, ideally, to promote tissue repair.

Primary Bone Cancer and Metastatic Bone Disease. Bone cancer occursinfrequently while bone metastases are present in a wide range ofcancers, including thyroid, kidney, and lung. Metastatic bone cancer isa chronic condition; survival from the time of diagnosis is variabledepending on tumor type. In prostate and breast cancer and in multiplemyeloma, survival time is measurable in years. For advanced lung cancer,it is measured in months. Cancer symptoms include pain, hypercalcemia,pathologic fracture, and spinal cord or nerve compression. Prognosis ofmetastatic bone cancer is influenced by primary tumor site, presence ofextra-osseous disease, and the extent and tempo of the bone disease.Bone cancer/metastasis progression is determined by imaging tests andmeasurement of bone specific markers. Recent investigations show astrong correlation between the rate of bone resorption and clinicaloutcome, both in terms of disease progression or death.

Multiple Myeloma. Multiple myeloma (MM) is a B-lymphocyte malignancycharacterized by the accumulation of malignant clonal plasma cells inthe bone marrow. The clinical manifestations of the disease are due tothe replacement of normal bone marrow components by abnormal plasmacells, with subsequent overproduction of a monoclonal immunoglobulin (Mprotein or M component), bone destruction, bone pain, anemia,hypercalcemia and renal dysfunction.

As distinct from other cancers that spread to the bone (e.g., breast,lung, thyroid, kidney, prostate), myeloma bone disease (MBD) is not ametastatic disease. Rather, myeloma cells are derived from the B-cellsof the immune system that normally reside in the bone marrow and aretherefore intimately associated with bone. Indeed, the bone marrowmicroenvironment plays an important role in the growth, survival andresistance to chemotherapy of the myeloma cells, which, in turn,regulate the increased bone loss associated with this disorder(world-wide-web at multiplemyeloma.org). Over 90% of myeloma patientshave bone involvement, versus 40-60% of cancer patients who have bonemetastasis, and over 80% have intractable bone pain. Additionally,approximately 30% of myeloma patients have hypercalcemia that is aresult of the increased osteolytic activity associated with this disease(Cavo et al., 2006).

Common problems in myeloma are weakness, confusion and fatigue due tohypercalcemia. Headache, visual changes and retinopathy may be theresult of hyperviscosity of the blood depending on the properties of theparaprotein. Finally, there may be radicular pain, loss of bowel orbladder control (due to involvement of spinal cord leading to cordcompression) or carpal tunnel syndrome and other neuropathies (due toinfiltration of peripheral nerves by amyloid). It may give rise toparaplegia in late presenting cases.

Myeloma Bone Disease. As discussed above, unlike the osteolysisassociated with other bone tumors, the MBD lesions are unique in thatthey do not heal or repair, despite the patients' having many years ofcomplete remission. Mechanistically, this seems to be related to theinhibition and/or loss of the bone-forming osteoblast during diseaseprogression. Indeed, bone marker studies and histomorphometry indicatethat both the bone-resorbing osteoclast and osteoblast activity areincreased, but balanced early in the disease, whereas overt MBD showshigh osteoclast activity and low osteoblast activity. Thus, MBD is adisorder in which bone formation and bone loss are uncoupled and wouldbenefit from therapies that both stimulate bone formation and retard itsloss.

A number of therapeutic approaches have been used in MBD, with theendpoints of treating pain, hypercalcemia, or the reduction of skeletalrelated events (SRE). Many of these may present serious complications.Surgery, such as vertebroplasty or kyphoplasty, that is performed forstability and pain relief has the attendant surgical risks (e.g.,infection) made worse by a compromised immune system and does notreverse existing skeletal defects. Radiation therapy and radioisotopetherapy are both used to prevent/control disease progression and havethe typical risks of irradiation therapies. More recently, drugs such asthe bisphosphonates that inhibit osteoclast activity have become astandard of therapy for MBD, despite the fact that they work poorly inthis disorder. In 9 major double-blind, placebo-controlled trials onbisphosphonates, only 66% of patients showed an effective reduction inpain; 56% showed a reduction in SRE and only 1 of the 9 demonstrated asurvival benefit.

B. Combination Treatments

As discussed, the present invention provides for the treatment of bonesdisease and bone trauma by stimulating the production of new bonetissue. Other agents may be used in combination with the anti-TGF-βantibodies of the present invention. More generally, these agents wouldbe provided in a combined amount (along with the anti-TGF-β antibodies)to produce any of the effects discussed above. This process may involvecontacting the cell or subject with both agents at the same time. Thismay be achieved by contacting the cell with a single composition orpharmacological formulation that includes both agents, or by contactingthe cell or subject with two distinct compositions or formulations, atthe same time, wherein one composition includes the intracellularinhibitor and the other includes the second agent.

Alternatively, one agent may precede or follow the other by intervalsranging from minutes to weeks. In embodiments where the agents areapplied separately to the cell or subject, one would generally ensurethat a significant period of time did not expire between the time ofeach delivery, such that the agents would still be able to exert anadvantageously combined effect on the cell or subject. In suchinstances, it is contemplated that one may contact the cell or subjectwith both modalities within about 12-24 h of each other and, morepreferably, within about 6-12 h of each other. In some situations, itmay be desirable to extend the time period for treatment significantly,however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2,3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

Various combinations may be employed, the anti-TGF-β antibody is “A” andthe other agent is “B”:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/BA/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/AA/A/B/AAdministration protocols and formulation of such agents will generallyfollow those of standard pharmaceutical drugs, as discussed furtherbelow. Combination agents include bisphosphonates (Didronel™, Fosamax™and Actonel™), SERMs (Evista) or other hormone derivatives, andParathyroid Hormone (PTH) analogs.

IV. PHARMACEUTICAL FORMULATIONS AND DELIVERY

A. Compositions and Routes

Pharmaceutical compositions of the present invention comprise aneffective amount of one or more anti-TGF-β antibodies dissolved ordispersed in a pharmaceutically acceptable carrier. The phrases“pharmaceutical or pharmacologically acceptable” refer to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to an animal, such as, forexample, a human, as appropriate. The preparation of a pharmaceuticalcomposition that contains at least one anti-TGF-β antibody, andoptionally an additional active ingredient, will be known to those ofskill in the art in light of the present disclosure, as exemplified byRemington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company,1990, incorporated herein by reference. Moreover, for animal (e.g.,human) administration, it will be understood that preparations shouldmeet sterility, pyrogenicity, general safety and purity standards asrequired by FDA Office of Biological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, 1289-1329, incorporated herein byreference). Except insofar as any conventional carrier is incompatiblewith the active ingredient, its use in the pharmaceutical compositionsis contemplated.

The anti-TGF-β antibody may be admixed with different types of carriersdepending on whether it is to be administered orally or by injection.The present invention can be administered buccally, intravenously,intradermally, transdermally, intrathecally, intraarterially,intraperitoneally, intranasally, intravaginally, intrarectally,topically, intramuscularly, intratumorally, into tumor vasculature,subcutaneously, mucosally, orally, topically, locally, inhalation (e.g.,aerosol inhalation), injection, infusion, continuous infusion, localizedperfusion bathing target cells directly, via a catheter, via a lavage,in cremes, in lipid compositions (e.g., nanoparticles, liposomes), or byother method or any combination of the forgoing as would be known to oneof ordinary skill in the art (see, for example, Remington'sPharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990,incorporated herein by reference). In particular, the anti-TGF-βantibody is formulated into a syringeable composition for use inintravenous admiminstration.

The anti-TGF-β antibody may be formulated into a composition in a freebase, neutral or salt form or ester. It may also besynthesized/formulated in a prodrug form. Pharmaceutically acceptablesalts, include the acid addition salts, e.g., those formed with the freeamino groups of a proteinaceous composition, or which are formed withinorganic acids such as for example, hydrochloric or phosphoric acids,or such organic acids as acetic, oxalic, tartaric, fumaric, or mandelicacid. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as for example, sodium, potassium, ammonium,calcium or ferric hydroxides; or such organic bases as isopropylamine,trimethylamine, histidine or procaine. Upon formulation, solutions willbe administered in a manner compatible with the dosage formulation andin such amount as is therapeutically effective.

Further in accordance with the present invention, the composition of thepresent invention suitable for administration is provided in apharmaceutically acceptable carrier with or without an inert diluent.The carrier should be assimilable and includes liquid, semi-solid, i.e.,pastes, or solid carriers. Except insofar as any conventional media,agent, diluent or carrier is detrimental to the recipient or to thetherapeutic effectiveness of the composition contained therein, its usein administrable composition for use in practicing the methods of thepresent invention is appropriate. Examples of carriers or diluentsinclude fats, oils, water, saline solutions, lipids, liposomes, resins,binders, fillers and the like, or combinations thereof. The compositionmay also comprise various antioxidants to retard oxidation of one ormore component. Additionally, the prevention of the action ofmicroorganisms can be brought about by preservatives such as variousantibacterial and antifungal agents, including but not limited toparabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol,sorbic acid, thimerosal or combinations thereof.

In a specific embodiment of the present invention, the composition iscombined or mixed thoroughly with a semi-solid or solid carrier. Themixing can be carried out in any convenient manner such as grinding.Stabilizing agents can be also added in the mixing process in order toprotect the composition from loss of therapeutic activity, i.e.,denaturation in the stomach. Examples of stabilizers for use in thecomposition include buffers, amino acids such as glycine and lysine,carbohydrates such as dextrose, mannose, galactose, fructose, lactose,sucrose, maltose, sorbitol, mannitol, etc.

In further embodiments, the present invention may concern the use of apharmaceutical lipid vehicle compositions that include anti-TGF-βantibodies, one or more lipids, and an aqueous solvent. As used herein,the term “lipid” will be defined to include any of a broad range ofsubstances that is characteristically insoluble in water and extractablewith an organic solvent. This broad class of compounds are well known tothose of skill in the art, and as the term “lipid” is used herein, it isnot limited to any particular structure. Examples include compoundswhich contain long-chain aliphatic hydrocarbons and their derivatives. Alipid may be naturally-occurring or synthetic (i.e., designed orproduced by man). Lipids are well known in the art, and include forexample, neutral fats, phospholipids, phosphoglycerides, steroids,terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides,lipids with ether and ester-linked fatty acids and polymerizable lipids,and combinations thereof.

One of ordinary skill in the art would be familiar with the range oftechniques that can be employed for dispersing a composition in a lipidvehicle. For example, the anti-TGF-β antibodies may be dispersed in asolution containing a lipid, dissolved with a lipid, emulsified with alipid, mixed with a lipid, combined with a lipid, covalently bonded to alipid, contained as a suspension in a lipid, contained or complexed witha micelle or liposome, or otherwise associated with a lipid or lipidstructure by any means known to those of ordinary skill in the art. Thedispersion may or may not result in the formation of liposomes.

The actual dosage amount of a composition of the present inventionadministered to an animal patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration. Depending upon the dosage and the route ofadministration, the number of administrations of a preferred dosageand/or an effective amount may vary according to the response of thesubject. The practitioner responsible for administration will, in anyevent, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

In certain embodiments, anti-TGF-β antibody pharmaceutical compositionsmay comprise, for example, at least about 0.1% of the antagonist, about0.5% of the antagonist, or about 1.0% of the antagonist. In otherembodiments, the antagonist may comprise between about 2% to about 75%of the weight of the unit, or between about 25% to about 60%, forexample, and any range derivable therein. Naturally, the amount of theantagonist in each therapeutically useful composition may be prepared issuch a way that a suitable dosage will be obtained in any given unitdose of the compound. Factors such as solubility, bioavailability,biological half-life, route of administration, product shelf life, aswell as other pharmacological considerations will be contemplated by oneskilled in the art of preparing such pharmaceutical formulations, and assuch, a variety of dosages and treatment regimens may be desirable.

In other non-limiting examples, a dose of anti-TGF-β antibodies may alsocomprise from about 0.1 microgram/kg/body weight, about 0.2microgram/kg/body weight, about 0.5 microgram/kg/body weight, about 1microgram/kg/body weight, about 5 microgram/kg/body weight, about 10microgram/kg/body weight, about 50 microgram/kg/body weight, about 100microgram/kg/body weight, about 200 microgram/kg/body weight, about 350microgram/kg/body weight, about 500 microgram/kg/body weight, about 1milligram/kg/body weight, about 5 milligram/kg/body weight, about 10milligram/kg/body weight, about 50 milligram/kg/body weight, about 100milligram/kg/body weight, about 200 milligram/kg/body weight, about 350milligram/kg/body weight, about 500 milligram/kg/body weight, to about1000 mg/kg/body weight or more per administration, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 5 mg/kg/body weight to about100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500milligram/kg/body weight, etc., can be administered, based on thenumbers described above.

In particular embodiments of the present invention, the anti-TGF-βantibodies are formulated to be administered via an alimentary route.Alimentary routes include all possible routes of administration in whichthe composition is in direct contact with the alimentary tract.Specifically, the pharmaceutical compositions disclosed herein may beadministered orally, buccally, rectally, or sublingually. As such, thesecompositions may be formulated with an inert diluent or with anassimilable edible carrier, or they may be enclosed in hard- orsoft-shell gelatin capsule, or they may be compressed into tablets, orthey may be incorporated directly with the food of the diet.

In certain embodiments, the active compounds may be incorporated withexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like(Mathiowitz et al., 1997; Hwang et al., 1998; U.S. Pat. Nos. 5,641,515,5,580,579 and 5,792,451, each specifically incorporated herein byreference in its entirety). The tablets, troches, pills, capsules andthe like may also contain the following: a binder, such as, for example,gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; anexcipient, such as, for example, dicalcium phosphate, mannitol, lactose,starch, magnesium stearate, sodium saccharine, cellulose, magnesiumcarbonate or combinations thereof; a disintegrating agent, such as, forexample, corn starch, potato starch, alginic acid or combinationsthereof; a lubricant, such as, for example, magnesium stearate; asweetening agent, such as, for example, sucrose, lactose, saccharin orcombinations thereof; a flavoring agent, such as, for examplepeppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.When the dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar, or both. When the dosage form is a capsule, it maycontain, in addition to materials of the above type, carriers such as aliquid carrier. Gelatin capsules, tablets, or pills may be entericallycoated. Enteric coatings prevent denaturation of the composition in thestomach or upper bowel where the pH is acidic. See, e.g., U.S. Pat. No.5,629,001. Upon reaching the small intestines, the basic pH thereindissolves the coating and permits the composition to be released andabsorbed by specialized cells, e.g., epithelial enterocytes and Peyer'spatch M cells. A syrup of elixir may contain the active compound sucroseas a sweetening agent methyl and propylparabens as preservatives, a dyeand flavoring, such as cherry or orange flavor. Of course, any materialused in preparing any dosage unit form should be pharmaceutically pureand substantially non-toxic in the amounts employed. In addition, theactive compounds may be incorporated into sustained-release preparationand formulations.

For oral administration, such as in the treatment of periodontaldisease, the compositions of the present invention may alternatively beincorporated with one or more excipients in the form of a mouthwash,dentifrice, buccal tablet, oral spray, gel or sublingualorally-administered formulation. For example, a mouthwash may beprepared incorporating the active ingredient in the required amount inan appropriate solvent, such as a sodium borate solution (Dobell'sSolution). Alternatively, the active ingredient may be incorporated intoan oral solution such as one containing sodium borate, glycerin andpotassium bicarbonate, or dispersed in a dentifrice, or added in atherapeutically-effective amount to a composition that may includewater, binders, abrasives, flavoring agents, foaming agents, andhumectants. Alternatively the compositions may be fashioned into atablet, gel or solution form that may be placed under the tongue, alongthe gum line, brushed on to teeth surfaces, or otherwise dissolved inthe mouth. U.S. Pat. Nos. 6,074,674 and 6,270,750, both incorporated byreference, describe topical, sustained release compositions forperiodontal procedures.

In further embodiments, anti-TGF-β antibodies may be administered via aparenteral route. As used herein, the term “parenteral” includes routesthat bypass the alimentary tract. Specifically, the pharmaceuticalcompositions disclosed herein may be administered for example, but notlimited to intravenously, intradermally, intramuscularly,intraarterially, intrathecally, subcutaneous, or intraperitoneally U.S.Pat. Nos. 6,537,514, 6,613,308, 5,466,468, 5,543,158; 5,641,515; and5,399,363 (each specifically incorporated herein by reference in itsentirety). Solutions of the active compounds as free base orpharmacologically acceptable salts may be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions mayalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms. The pharmaceutical forms suitable for injectable useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form must be sterile andmust be fluid to the extent that easy injectability exists. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (i.e., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it may be desirable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, and intraperitoneal administration. In thisconnection, sterile aqueous media that can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics standards.

Sustained release formulations for treating of bone conditions includeU.S. Pat. Nos. 4,722,948, 4,843,112, 4,975,526, 5,085,861, 5,162,114,5,741,796 and 6,936,270, all of which are incorporated by reference.Methods and injectable compositions for bone repair are described inU.S. Pat. Nos. 4,863,732, 5,531,791, 5,840,290, 6,281,195, 6,288,043,6,485,754, 6,662,805 and 7,008,433, all of which are incorporated byreference.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. A powdered composition is combined with a liquidcarrier such as, e.g., water or a saline solution, with or without astabilizing agent.

B. Devices

In addition to providing anti-TGF-β antibodies for administration byroutes discussed above, such agents, alone or in combination, maybe usedin the context of devices, such as implants. A variety of bone relatedimplants are contemplated, including dental implants, joint implantssuch as hips, knees, and elbows, vertebral/spinal implants, and others.The anti-TGF-β antibodies may be impregnated in a surface of theimplant, including in a bioactive matrix or coating. The inhibitor maybe further formulated to sustained, delayed, prolonged or time release.The coating may comprise polymers, for example, such as those listedbelow. The following is a list of U.S. patents relating to bone implantsand devices which may be utilized in accordance with this embodiment ofthe invention:

TABLE 1 BONE IMPLANT PATENTS U.S. Patent* Patent Title 7,044,972 Boneimplant, in particular, an inter-vertebral implant 7,022,137 Bonehemi-lumbar interbody spinal fusion implant having an asymmetricalleading end and method of installation thereof 7,001,551 Method offorming a composite bone material implant 6,994,726 Dual functionprosthetic bone implant and method for preparing the same 6,989,031Hemi-interbody spinal implant manufactured from a major long bone ringor a bone composite 6,988,015 Bone implant 6,981,975 Method forinserting a spinal fusion implant having deployable bone engagingprojections 6,981,872 Bone implant method of implanting, and kit for usein making implants, particularly useful with respect to dental implants6,929,662 End member for a bone fusion implant 6,923,830 Spinal fusionimplant having deployable bone engaging projections 6,921,264 Implant tobe implanted in bone tissue or in bone tissue supplemented with bonesubstitute material 6,918,766 Method, arrangement and use of an implantfor ensuring delivery of bioactive substance to the bone and/or tissuesurrounding the implant 6,913,621 Flexible implant using partiallydemineralized bone 6,899,734 Modular implant for fusing adjacent bonestructure 6,860,884 Implant for bone connector 6,852,129 Adjustable bonefusion implant and method 6,802,845 Implant for bone connector 6,786,908Bone fracture support implant with non-metal spacers 6,767,367 Spinalfusion implant having deployable bone engaging projections 6,761,738Reinforced molded implant formed of cortical bone 6,755,832 Bone plateimplant 6,730,129 Implant for application in bone, method for producingsuch an implant, and use of such an implant 6,689,167 Method of usingspinal fusion device, bone joining implant, and vertebral fusion implant6,689,136 Implant for fixing two bone fragments to each other 6,666,890Bone hemi-lumbar interbody spinal implant having an asymmetrical leadingend and method of installation thereof 6,652,592 Segmentallydemineralized bone implant 6,648,917 Adjustable bone fusion implant andmethod 6,607,557 Artificial bone graft implant 6,599,322 Method forproducing undercut micro recesses in a surface, a surgical implant madethereby, and method for fixing an implant to bone 6,562,074 Adjustablebone fusion implant and method 6,562,073 Spinal bone implant D473,944Bone implant 6,540,770 Reversible fixation device for securing animplant in bone 6,537,277 Implant for fixing a bone plate 6,506,051 Boneimplant with intermediate member and expanding assembly 6,478,825Implant, method of making same and use of the implant for the treatmentof bone defects 6,458,136 Orthopaedic instrument for sizing implantsites and for pressurizing bone cement and a method for using the same6,447,545 Self-aligning bone implant 6,436,146 Implant for treatingailments of a joint or a bone 6,371,986 Spinal fusion device, bonejoining implant, and vertebral fusion implant 6,370,418 Device andmethod for measuring the position of a bone implant 6,364,880 Spinalimplant with bone screws 6,350,283 Bone hemi-lumbar interbody spinalimplant having an asymmetrical leading end and method of installationthereof 6,350,126 Bone implant 6,287,343 Threaded spinal implant withbone ingrowth openings 6,270,346 Dental implant for bone regrowth6,248,109 Implant for interconnecting two bone fragments 6,217,617 Boneimplant and method of securing 6,214,050 Expandable implant forinter-bone stabilization and adapted to extrude osteogenic material, anda method of stabilizing bones while extruding osteogenic material6,213,775 Method of fastening an implant to a bone and an implanttherefor 6,206,923 Flexible implant using partially demineralized bone6,203,545 Implant for fixing bone fragments after an osteotomy 6,149,689Implant as bone replacement 6,149,688 Artificial bone graft implant6,149,686 Threaded spinal implant with bone ingrowth openings 6,126,662Bone implant 6,083,264 Implant material for replacing or augmentingliving bone tissue involving thermoplastic syntactic foam 6,058,590Apparatus and methods for embedding a biocompatible material in apolymer bone implant 6,018,094 Implant and insert assembly for bone anduses thereof 5,976,147 Modular instrumentation for bone preparation andimplant trial reduction of orthopedic implants 5,906,488 Releasableholding device preventing undesirable rotation during tightening of ascrew connection in a bone anchored implant 5,899,939 Bone-derivedimplant for load-supporting applications 5,895,425 Bone implant5,890,902 Implant bone locking mechanism and artificial periodontalligament system 5,885,287 Self-tapping interbody bone implant 5,819,748Implant for use in bone surgery 5,810,589 Dental implant abutmentcombination that reduces crestal bone stress 5,759,035 Bone fusiondental implant with hybrid anchor 5,720,750 Device for the preparationof a tubular bone for the insertion of an implant shaft 5,709,683Interbody bone implant having conjoining stabilization features for bonyfusion 5,709,547 Dental implant for anchorage in cortical bone 5,674,725Implant materials having a phosphatase and an organophosphorus compoundfor in vivo mineralization of bone 5,658,338 Prosthetic modular bonefixation mantle and implant system D381,080 Combined metallic skull basesurgical implant and bone flap fixation plate 5,639,402 Method forfabricating artificial bone implant green parts 5,624,462 Bone implantand method of securing D378,314 Bone spinal implant 5,607,430 Bonestabilization implant having a bone plate portion with integral cableclamping means 5,571,185 Process for the production of a bone implantand a bone implant produced thereby 5,456,723 Metallic implantanchorable to bone tissue for replacing a broken or diseased bone5,441,538 Bone implant and method of securing 5,405,388 Bone biopsyimplant 5,397,358 Bone implant 5,383,935 Prosthetic implant withself-generated current for early fixation in skeletal bone 5,364,268Method for installing a dental implant fixture in cortical bone5,312,256 Dental implant for vertical penetration, adapted to differentdegrees of hardness of the bone *The preceding patents are all herebyincorporated by reference in their entirety.

V. SCREENING ASSAYS

In still further embodiments, the present invention provides methodsidentifying new and useful antibodies against TGF-β for use instimulating bone production. For example, a method generally comprises:

-   -   (a) providing a candidate antibody;    -   (b) admixing the candidate modulator with a cell or a suitable        experimental animal;    -   (c) measuring osteoblast or osteoclast activity, or bone growth,        strength, mass or formation; and    -   (d) comparing the characteristic measured in step (c) with that        observed in the absence of the candidate,    -   wherein a difference between the measured characteristic        indicates that said candidate is, indeed, a bone production        stimulator.

Assays may be conducted in isolated cells or in organisms includingtransgenic animals. Bone formation can be identified by the von Kossa orAlzarin Red stains, FTIR or Raman spectrometric analysis, or byfluorochromes linked to compounds that bind bone.

It will, of course, be understood that all the screening methods of thepresent invention are useful in themselves notwithstanding the fact thateffective candidates may not be found. The invention provides methodsfor screening for such candidates, not solely methods of finding them.

VI. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Materials & Methods

Antibodies. The 1D11 antibody was generated by Genzyme Corporation(Framingham). Control antibody (13C4) consistent of an identical IgGcomplex lacking any TGF-β binding capabilities.

Treatment regimen. Normal 13-week old male C57B1/6 mice (Harlan) (n=5)were treated with 10 mg/kg/×3 week of 1D11 or control antibody. Eachreagent was administered by sterile intra-peritoneal injection over a 4week time period (FIG. 1). All animal procedures were conducted inaccordance with IACUC protocols approved by Vanderbilt UniversityMedical Center.

Imaging. Tibia and femure were analyzed by μCT scanning (μCT40, Scanco)at an isotropic voxel size of 12 μm (55 Kv). After the growth plate wasidentified in each scan set, the metaphyseal region 200 μm below thisarea was scanned and analyzed for alterations in trabecular boneparameters (Threshold 280).

Histology and histomorphometry. Lumbar vertebral bodies (L3-5) and longbones were collected following sacrifice and fixed for up to 48 hrs in10% formalin. Undecalcified regions of vertebrae were processed andembedded in a methylmethacrylate-based resin and sectioned at 5 μm.Sections were deplasticized and stained for bound calcium ions using theVon Kossa procedure with a van Gieson counterstain, or using apost-coupling staining technique for tartrate-resistant acid phosphatase(TRAP). Long bones were decalcified for 2 weeks in 10% EDTA andprocessed to paraffin wax. Samples were sectioned at 5 μm and statedwith H&E/Orange G, or for TRAP activity. Bone volume, cellulardistribution was quantified histomorphometrically using Osteomeasurequantification software (Osteometrics).

Gene expression. The RANKL/OPG gene expression ratio was assessed in1D11- or control-treated T23 osteoblast cells, with RNA isolated usingRNEasy extraction kits (Qiagen). Validated Taqman primers were purchasesand samples analyzed using a 7300 Real Time PCR system (AppliedBiosystems) under conditions recommended by the manufacturer.Osteogeneic expression was assessed using standard RT-PCR techniques.

Protein expression. Normal serum was isolated from treated and untreatedmice by exsanguination prior to sacrifice. Serum was assesssed by enzymelinked immunosorbent assay (ELISA) for levels of soluble RANKL or OPGprotein using the Quantikine Immunoassay system (R&D Systems) withconcentrate and ×5 serum dilution used for RANKL and OPG, respectively.

Urine resporption assay. Urine samples were collected from all animalsprior to sacrifice. The collagen breakdown product deoxypyridinoline(DPD) was quantified by ELISA using the MicroVue-DPD assay (QuidelCorp.) following manufacturer's guidelines and normalized to urinarycreatinine levels (MicroVue-Creatinine, Quidel Corp.).

Biochemical testing. The strength and modulus of the diaphyseal regionof the femur were analyzed biochemically. Fresh femurs were horizontallypositioned on support rollers and monotonically loaded on three-pointbending at a rate of 3 mm/min, using a material testing system(Dynamight 8841; Instron). The force displacement curve was recorded toprovide the maximum force endured by the bone an the initial stiffness.Using uCT-derived moment of inertia and flexural equations from beamtheory (Schriefer et al., 2005), the inventors converted thesestructural properties to whole bone bending strength and modulus.

Raman microspectroscopy. The chemical composition of the bone tissue wascharacterized by confocal Raman microspectroscopy (Renishaw). Tibia wereembedded in PMMA and cut at the metaphysis below the growth place toexpose a cross-section of the cortex. This surface was ground onsuccessive grits of silicon carbide paper and polished with 1 μm aluminaslurry. A 50× objective focuses the laser (785 nm laser diode source) toa 3 μm region below the surface of the tissue, and inelastic light wascollected by a Renishaw spectrograph (1 cm⁻¹ spectral resolution). Themeasured spectra consisted of three accumulations with an integrationtime of 10 s each. Using custom Matlab scripts, background fluorescencein the spectra was subtraced by a modified polynomial fitting algorithm(Lieber et al., 2003). Spectra were collected from 10 trabecularlocations with the tibial metaphysis (ground below the growth plate).Mineral-to-collagen ratio was calculated as the v1 phosphate peakintensity (962 cm⁻¹) per proline peak intensity (856 cm⁻¹) and averagedper bone.

Nanoindentation. Modulus at the tissue level was quantified bynanoindentation. Resin-embedded regions of the tibial diaphysis wereprobed using a Nanoidenter XP (MTS XP). A Berkovitch diamond tip(inclination angle: 142.3°; radius: 100 nm) was pressed into the surfaceusing trapezoidal loading scheme as follows: 1) load at a strain rate of0.5/s to a depth of 1 μm, 2) hold at P_(max) for 10 seconds, 3) unloadat 350 μN s⁻¹ to 90% of P_(max), and 4) leave the indenter on thesurface for 60 seconds in order to establish the thermal drift. From theresulting force-displacement curve, the elastic modulus (E) of thetissue at the point of indenting (0.25 μm resolution) was calculatedfollowing the methods of Oliver and Pharr (2004). This involves aninitial calibration procedure using fused silica to establish therelationship between depth of indent and contact area of the tip, and todetermine the slope of the unloading portion of the force-displacementcurve. Ten indents were collected per bone, with data represented asmean±SE.

Statistical analysis. Statistically significant values were determinedby the Mann-Whitney and Students t-test with p-values less than 0.05considered significant.

Example 2 Results

Inhibition of TGF-β by 1D11 antibody treatment, outlined in FIG. 1,significantly increased long bone volume compared to controls (FIGS.2A-B). Trabecular bone at the tibial metaphysis analyzed by μCT scanningshowed a dramatic increase in overall BV/TV (FIG. 2C), bone mineraldensity (BMD) (FIG. 2D), trabecular thickness (FIG. 2F) and decreasedtrabecular separation (FIG. 2F) in animals treated with 1D11 compared tocontrol-treated mice. Histomorphometric analysis of undecalcifiedsections from lumbar vertebra supported μCT analysis of long bones.1D11-mediated TGF-β inhibition led to a 54% increase in trabecularBV/TV. This increase in bone was accompanied by greater trabecularnumber, decreased trabecular separation and increased trabecularthickness (FIGS. 3A-B).

An analysis of bone cell distribution in TRAP-stained vertebral sectionsshowed significantly reduced osteoclast numbers and surface areafollowing 1D11 treatment (FIGS. 4A-C). In contrast, elevated osteoblastnumbers and osteoblast area lining the bone surface in mice receiving1D11 was observed, compared to controls (FIG. 4D). In addition toalterations in bone cell numbers, overall bone mass and skeletalintegrity can also be affected by changes in the remodelling rate ofbone. To determine bone turnover rates following treatment with 1D11,the inventors assessed the collagen breakdown product deoxypyridinoline(DPD) in urine samples collected at sacrifice. In support ofhistomorphometric data, the DPD/creatinine ratio indicated a decrease inresportive activity following treatment with 1D11 (control=39.3±5.6;1D11=12.0±6.2 μg protein).

It has become very clear over recent years that in addition to theoverall quantity of bone present within the skeleton, the quality ofbone is also an essential element to be considered when analyzing newtherapeutics and their effects on the skeleton. To address this, theinventors performed 3-point bending on excised femurs to determine theeffect of 1D11 treatment on the biomechanical properties of bone.Blocking TGF-β signaling resulted in considerably stronger bones withincreased whole bone bending strength and modulus (FIG. 5). The effectof 1D11 on skeletal modulus was also examined at a tissue level usingnanoindentation. These findings support biomechanical data showingenhanced tissue-level modulus following treatment with 1D11 (Table 2).In addition, Rama microspectroscopy allowed the inventors to analyze andquantify the effect of TGF-β blockade on the compositional components ofbone. These studies demonstrated an 11% increase in mineral-to-collagentratio of trabecular bone in the tibial methaphysis following treatmentwith 1D11. However, the quality of hydroxyapatite and overallcrystallinity of the inorganic component remained unchanged (Table 2).

Skeletal integrity is maintained when osteoblast and osteoclastformation an activity are balanced. A primary mechanism mediatingosteoclastic bone resporption occurs via RANKL/OPG expressionosteoclasts. As TGF-β has previously been shown to alter the RANKL/OPGratio) (Mohammad et al., 2009; Karst et al., 2004; Quinn et al., 2001;Thirunavukkarasu et al., 2001), the inventors examined the effect oftreatment with 1D11 on RANKL/OPG gene expression in osteobalst celllines in vitro and assessed RANKL and OPG prtoein levels in vivo, inserum samples from 1D11- or control-treated animals. In culture, TGF-βtreatment decreased RANKL mRNA expression (rank1/gapdh; 1.6±0.2 versus0.7±0.1) and increased OPG gene expression (opg/gapdh: 1.7±0.1 versus2.4±0.2). This effect was blocked by the addition of 1D11 to osteobalstcultures. However, no significant changed in individual RANKL(control=31.9±7.6; 1D11=17.0±1.8 pg/ml) or OPG (control=487.7±22.6;1D11=451.5±20.4 pg/ml) protein levels were detectable in serum samplesfrom treated or untreated mice, although the reduced RANKL levels didlead to a 50% decrease in the overall RANKL/OPG ratio. In addition, thedirect effect of TGF-β on osteogenic gene expression was analysis invitro. Treatment of 2T3 osteoblast cells with TGF-β induced a 49.3%decrease in alkaline phosphatase gene expression and a 331.8% increasein PTHrP expression, as determined by PCR. This increase was completelyprevented by treatment with 1D11 in addition to TGF-β. No alterationswere observed in the expression levels of runx2, β-catenin, type 1collagent or osteocalcin, with identical results observed in the MC3T3osteoblast cell line. Together, these investigations confirm an overallbeneficial effect on the skeleton following the neutralization of TGF-βin the bone marrow environment.

TABLE 2 Compositional parameters of treated bone Control 1D11Nanoindentation Tissue-level modulus (GPa) 22.7 ± 0.5  24.3 ± 1.3* Ramanmicrospectroscopy Mineral/collagen ratio (%) 5.5 ± 0.1  6.1 ± 0.1*Carbonate substitutions 0.185 ± 0.012 0.169 ± 0.009 Crystallinity 0.078± 0.005 0.081 ± 0.002

Example 3 Discussion

This study investigates the use of a TGF-β neutralizing antibody as ananabolic bone agent and highlights the potential of TGF-β inhibition asa mechanism to increase bone mass. The inventors employed standard,accepted techniques along with emerging technologies to thoroughlyanalyze bone volume, density, strength and composition. Together, thesestudies demonstrate that drugs aimed at blocking the TGF-β signalingpathway have the capacity to positively regulate osteoblast numberswhile simultaneously decreasing the amount of active osteoclasts in themarrow. This results in a profound increase in bone volume and quality,similar to that seen in PTH-treated rodent studies (Dempster et al.,1993).

There is a considerable need for more efficacious bone anabolic agents.Currently, the major therapeutic approach to excessive bone loss isthrough the use of anti-resorptives such as bisphosphonates. While theseagents are certainly capable of repressing further bone resorption, theyare unable to stimulate new cycles of formation to replace the bonewhich has been lost. The inventors have shown that targeting TGF-β witha neutralizing antibody has the ability to prevent bone destruction bydecreasing osteoclasts, while simultaneously increasing osteoblasts. Thedirect result is a net improvement in bone mass within the appendicularand axial skeletal regions. Long bones were also shown to beconsiderably stronger with enhanced matrix compositional propertiesfavorable to normal skeletal function.

Elevated bone loss, such as that seen in osteoporosis, frequently leadsto an increase in fracture risk. This feature ultimately results from anoverall loss of strength and decreased bone quality within the skeleton.It has been suggested that this deficit in bone quality cannot beaccounted for by the decrease in bone volume alone, suggesting anintrinsic defect in the production of new bone matrix in theseindividuals. Normal bone typically comprises of a balanced ratio oforganic collagen matrix and inorganic mineral component. While excessivedysregulation of each element results in profound skeletal defects, theratio can be modified throughout life to improve the overall strength ofbone and resistance to fracture. Treatment with 1D11 increased themineral to collagen ratio of trabecular bone without impairing thepurity of the hydroxyapatite, as assessed by the level of carbonatesubstitutions within the crystal, suggesting that TGF-β inhibitionwithin this environment favors the production of improved quality bone.Also, the enhanced compositional features of 1D11-treated bone,translate well to an overall increase in bone strength in these animals.This data is supported by genetically modified mouse models wheredisrupted TGF-β signaling increases bone strength (Balooch et al.,2005), though it was not known whether this system would represent aviable and effective therapeutic approach to improve bone mass. Thedramatic beneficial effects reported in our study provide strongevidence for the development of pharmaceutical agents specificallytargeted at TGF-β inhibition to enhance bone mass and strength.

Intermittent doses of parathyroid hormone (PTH) currently represent theonly clinically available approach to increase bone volume. However, themechanism through which PTH induces this effect is still not clear.Moreover, continuous PTH treatment is proven to stimulate osteoclasticbone resorption and decrease overall bone mass (Raisz, 2005). Like PTH,neutralizing TGF-β with 1D11 antibody therapy vastly improved skeletalparameters, and like PTH the true mechanism through which this may beoccurring is still unknown. But in contrast to the effects of PTH, 1D11negatively regulates osteoclasts, inhibiting bone degradation andoffering a dual approach to enhance bone mass through increasedosteoblast numbers and decreased osteoclastic resorption.

Despite a wealth of literature describing TGF-β effects on bone cells,it remains unclear how TGF-β inhibition mediates skeletal events invivo. Recent studies suggest a dysregulation in primary osteoclastogenicmolecules (RANKL/OPG) or ephrin mediated bone remodeling (Mohammad etal., 2009).

The inventors examined soluble RANKL and OPG protein levels in mousesera following treatment with 1D11 or control and noted a trend toward adecrease in the RANKL/OPG ratio, brought about by decreased RANKLlevels. This finding may be associated with the decreased expression ofPTHrP observed in osteoblasts treated with 1D11, as PTHrP is known tostimulate RANKL in this cell type (Lee and Lorenzo, 1999; Itoh et al.,2000). While these in vivo observations are consistent with currentstudies (Mohammad et al., 2009) and suggest that 1D11 treatment couldincrease bone volume by suppressing TGF-β regulation of RANKL or OPG inosteoblasts, contrasting in vitro molecular studies, indicate that TGF-βtreatment leads to a significant reduction in RANKL and increase in OPGmRNA expression, which could be blocked by 1D11. These findings areconsistent with published studies documenting TGF-β regulation ofRANKL/OPG in vitro (Quinn et al., 2001; Thirunavukkarasu et al., 2001)and strongly suggest a diverse role of TGF-β in normal physiology, whichis not well-recapitulated in culture systems, and is likely dependent onthe interactions with other regulatory molecules. A carefulinterpretation of results is therefore necessary when analyzingdifferences between TGF-β function in vitro and in vivo.

Osteoblasts derive from a mesenchymal stem cell population within thebone marrow. Precursor cells are triggered to commit to the osteoblastlineage by stimulatory factors such as BMP-2 (Katagiri et al., 1990;Takuwa et al., 1991). Molecular analysis of osteogenic gene expression,suggested that osteoblast activity may be altered by 1D11-treatment invitro, evidenced by increased alkaline phosphatase levels. This findingcorrelates with published data (Filvaroff et al., 1999; Alliston et al.,2001; Kang et al., 2005), though fails to illustrate any major effect onthe maturation of precursors to increase osteoblast numbers, asdemonstrated by our in vivo findings. This suggests that local TGF-βinhibition in the bone marrow may influence bone formation during thistreatment period, but the true mechanism governing any change inosteoblastogenesis remains unknown.

In addition to direct effects on bone cells, the inventors are unable toexclude a systemic effect of TGF-β in this system. It is plausible thatTGF-β control of normal physiological processes negatively impact bonemass, and blocking these effects are ultimately beneficial for skeletalhealth. It is also possible that TGF-β control of bone cell formation oractivity varies temporally as the cellular population of the marrowchanges. This would be consistent with in vitro studies describingdifferential effects on precursor cells compared to mature matrixforming osteoblasts (Mundy and Bonewald, 1990). The inventors usedmature C57BI/6 male mice to highlight the beneficial effects of TGF-βblockade on the skeleton. Further studies using bone-loss models will benecessary to assess whether 1D11 treatment improves bone under theseconditions. Despite this, our use of C57BI/6 mice, which are reported tohave the lowest BMD of all available mouse strains (Beamer et al.,1996), suggests that TGF-β inhibition is likely to improve skeletalproperties in low bone mass, aged or osteopenic models also.

In support of this data, small molecules aimed at blocking TGF-βsignaling by inhibiting TGF-β receptor kinase activity have recentlybeen shown to increase bone mass (Mohammad et al., 2009). Although thesemolecules demonstrate a significant enhancement in trabecular bone,1D11-mediated TGF-β blockade increases both trabecular bone volume andimproves cortical bone strength. These superior skeletal effects may bea result of the complete abrogation of TGF-β signaling through the avidbinding and neutralization of all extracellular TGF-β isoforms, comparedto the inhibition of intracellular receptor associated enzymes currentlytargeted by small molecules. These findings clearly illustrate thepotential of compounds which can specifically target TGF-β in vivo, andsuggest a therapeutic approach to increase bone mass in conditions whereexcessive bone destruction is prevalent, such as cancer-induced bonedisease or osteoporosis.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

VII. REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. A method of increasing bone strength, bone mass, bone growth and/orvolume in a subject comprising: administering to said subject anantibody that binds immunologically the TGF-β.
 2. The method of claim 1,wherein said antibody binds all three isoforms of TGF-β.
 3. The methodof claim 1, wherein said antibody is designated as 1D11.
 4. The methodof claim 1, wherein said antibody is administered to said subjectsystemically.
 5. The method of claim 4, wherein said antibody isadministered intravenously, intra-peritoneally, intramuscularly,subcutaneously or topically.
 6. The method of claim 1, wherein saidantibody is administered to a bone target site.
 7. The method of claim6, wherein said antibody is injected at said site.
 8. The method ofclaim 6, wherein said antibody is comprised in a time-release deviceimplanted at said site.
 9. The method of claim 1, wherein said subjectis a human.
 10. The method of claim 1, wherein said subject is anon-human animal.
 11. The method of claim 10, wherein said non-humananimal is a mouse, a rat, a rabbit, a dog, a cat, a horse, a monkey or acow.
 12. The method of claim 1, wherein said subject has cancer.
 13. Themethod of claim 1, wherein said subject does not have cancer.
 14. Themethod of claim 1, further comprising at least a second administrationof said antibody.
 15. The method of claim 14, wherein said subjectreceive three administrations per week.
 16. The method of claim 14,wherein said subject receives at least 9 administrations.
 17. The methodof claim 1, further comprising assessing bone mass followingadministration of said antibody.
 18. The method of claim 17, whereinassessing comprises bone imaging.
 19. The method of claim 1, whereinsaid subject suffers from osteoporosis, bone fracture, bone loss due totrauma, or Paget's Disease.
 20. The method of claim 1, wherein saidsubject suffers from bone loss due to cancer metastasis. 21-29.(canceled)
 30. The method of claim 1, further comprising identifying apatient in need of increased bone mass and/or volume.
 31. A method ofincreasing osteoblast number and/or decreasing osteoclast number in asubject comprising administering to said subject an antibody that bindsimmunologically the TGF-β.
 32. (canceled)
 33. (canceled)
 34. A method ofdecreasing TGF-β signaling in a subject comprising administering to saidsubject an antibody that binds immunologically the TGF-β.